1. Field of the Invention
The present invention relates to laser capture microdissection. More particularly, the present invention relates to broadband absorbing films for use in laser capture microdissection (LCM).
2. The Prior Art
The LCM technique is generally described in the recently published article: Laser Capture Microdissection, Science, Volume 274, Number 5289, Issue 8, pp 998-1001, published in 1996, incorporated herein by reference. The purpose of the LCM technique is to provide a simple method for the procurement of selected human cells from a heterogeneous population contained ion a typical histopathology biopsy slide.
A typical tissue biopsy sample consists of a 5 to 10 micron slice of tissue that is placed on a glass microscope slide using techniques well known in the field of pathology. This tissue slice is a cross section of the body organ that is being studied. The tissue consists of a variety of different types of cells. Often a pathologist desires to remove only a small portion of the tissue for further analysis.
LCM employs a thermoplastic transfer film that is placed on top of the tissue sample. This film is manufactured containing organic dyes that are chosen to selectively absorb in the near infrared region of the spectrum overlapping the emission region of common AlGaAs laser diodes. When the film is exposed to the focused laser beam the exposed region is heated by the laser and melts, adhering to the tissue in the region that was exposed. The film is then lifted from the tissue and the selected portion of the tissue is removed with the film.
Thermoplastic transfer films such as a 100 micron thick ethyl vinyl acetate (EVA) film available from Electroseal Corporation of Pompton Lakes, N.J. (type E540) have been used in LCM applications. The film is chosen to have a low melting point of about 90 C.
The thermoplastic EVA films used in LCM techniques have been doped with dyes, such as an infrared napthalocyanine dye, available from Aldrich Chemical Company (dye number 43296-2 or 39317-7). These dyes have a strong absorption in the 800 nm region, a wavelength region that overlaps with laser emitters used to selectively melt the film. The dye is mixed with the melted bulk plastic at an elevated temperature. The dyed plastic is then manufactured into a film using standard film manufacturing techniques. The dye concentration in the plastic is about 0.001 M.
While the films employed in LCM applications have proved satisfactory for the task, they have several drawbacks. The optical absorption of a dye impregnated film is a function of its thickness. This property of the film may be in conflict with a desire to select film thickness for other reasons.
The organic dyes which are used to alter the absorption characteristics of the films may have detrimental photochemistry effects in some cases. This could result in contamination of LCM samples. In addition, the organic dyes employed to date are sensitive to the wavelength of the incident laser light and thus the film must be matched to the laser employed.
It is therefore an object of the invention to provide a film for LCM applications which overcomes the shortcomings of the prior art.
It is another object of the present invention to provide a simple LCM film to be used in an LCM instrument.
Yet another object of the present invention is to provide an LCM film which is very thin and can have very high optical absorption.
A further object of the present invention is to provide an optical quality LCM film which is not sensitive to the wavelength of the incident laser light.
A further object of the present invention is to provide optical quality LCM films of adjustable thickness.
Another object of the present invention is to provide an LCM film whose optical density can be adjusted so that sufficient light is transmitted through the sample to permit the sample to be viewed with back light illumination.
Yet another object of the present invention is to provide an LCM film which does not use an organic dye that might have detrimental photochemistry effects.
Yet another object of the present invention is to provide an LCM film which does not employ an organic dye which does not have to be dissolved into the EVA polymer at high concentrations.
A thermoplastic film for LCM tissue transfer is thermally coupled to a broadband energy-absorbing material. The broadband energy-absorbing material may either be introduced into the film composition as a dopant or may be in thermal contact with the film.
According to one embodiment of the present invention, a film for LCM tissue transfer comprises a layer of a transparent support film; a layer of a broadband absorbing film; and a layer of a low temperature thermoplastic; the absorbing film having an overall thickness chosen to absorb a desired fraction of incident laser light.
According to this embodiment of the invention, the film consists of a sandwich of layers containing a transparent support film such as mylar or polyester, a broadband absorbing film such as a metal film of nichrome or titanium, and a low temperature thermoplastic layer such as ethyl vinyl acetate (EVA). The metal film thickness is chosen so as to absorb a desired fraction of incident laser light. A typical film may absorb say 10% to 90% of the incident light. The metal thickness is adjusted to provide this optical density by controlling the length of time of the sputtering deposition process. The metal absorbing layer can be sandwiched between two layers of EVA if desired.
A method for fabricating a multilayer film for LCM tissue transfer comprises the steps of: providing a support layer; forming a thin layer of a broadband absorbing film onto the support layer, the broadband absorbing film having a thickness chosen to absorb a desired fraction of incident laser light; and coating the broadband absorbing film with a layer of thermoplastic.
According to the method of the present invention, one embodiment of the film is fabricated by evaporating a thin layer of metal film onto the polyester support using a deposition technique such as sputtering. The film thickness is chosen so as to absorb a desired fraction of incident laser light, usually between 10% and 95%. The metal thickness is adjusted to provide this optical density by controlling the length of time of the sputtering deposition process. The polyester/metal film is then coated with a thin layer (a few to 100 microns) of EVA thermoplastic using a spin coater to achieve a uniform layer across the surface. The spin coating process can be repeated several times to adjust the thickness of the EVA film.
According to one embodiment of the present invention, a thermoplastic film is doped with a broadband energy-absorbing material. The dopant concentration is adjusted so as to give a suitable absorption, usually between 10% and 95%, for the desired thickness of EVA film.
According to another embodiment of the present invention, a broadband absorbing material is deposited on a substrate such as a cap for a biological analysis vessel. The metal film is deposited onto the bottom of a cap and then the cap is coated with EVA.